[D-Trp6 ]-LH-RH and intermediates therefor

ABSTRACT

The decapeptide [D-Trp 6  ]-LH-RH, salts thereof, and intermediates used for the synthesis thereof are disclosed. The decapeptide has potent LH- and FSH-releasing hormone properties.

The invention described herein was made in the course of work under agrant or award from the Department of health, Education and Welfare.

BACKGROUND OF THE DISCLOSURE

a. Field of the Invention

This invention relates to the decapeptide(pyro)Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂, salts thereof andintermediates for the synthesis thereof.

The decapetide of this invention also is calledL-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolyglycinamideand may be designated by the abbreviation [D-Trp⁶ ]-LH-RH.

b. Background of the Invention

Luteinizing hormone (LH) and follicle-stimulating hormone (FSH) are bothgonadotrophic hormones elaborated by the pituitary gland of humans andof animals. LH together with FSH stimulates the release of estrogensfrom the maturing follicles in the ovary and induces the process ofovulation in the female. In the male LH stimulates the interstitialcells and is for that reason also called interstitial cell stimulatinghormone (ICSH). FSH induces maturation of the follicles in the ovary andtogether with LH, plays an important role in the cyclic phenomena in thefemale. FSH promotes the development of germinal cells in the testes ofthe male. Both LH and FSH are released from the pituitary gland by theaction of LH- and FSH-releasing hormone, and there is good evidence thatsaid releasing hormone is elaborated in the hypothalamus and reaches thepituitary glnd by a neurohumoral pathway, see e.g., A. V. Schally, etal., Recent Progress in Hormone Research, 24, 497 (1968).

The natural LH- and FSH-releasing hormone has been isolated from pighypothalami and its constitution elucidated by A. V. Schally, et al.,Biochem. Biophys. Res. Commun., 43 , 393 and 1334 (1971), who proposedthe decapeptide structure(pyro)-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂.

This constitution has been confirmed by synthesis; for example, see H.Matsuo, et al., Biochem. Biophys. Res. Comm., 45, 822 (1971) and R.Geiger et al., ibid, 45, 767 (1971).

Hereinafter the natural LH- and FSH-releasing hormone is called LH-RH.

Because of the importance of LH-RH to both diagnostic and therapeuticmedicine, considerable interest has been shown in the preparation of newcompounds having improved properties over the natural hormone. Oneapproach to this goal has been the replacement of an amino acid residueof LH-RH with another amino acid. Although in a few instancesdecapeptides containing such a replacement have been found to be moreactive than LH-RH, for example, [D-Ala⁶ ]-LH-RH, A. Arimura, et al.,Endocrinology, 95, 1174 (1971) and [D-Leu⁶ ]-LH-RH, J. A.Vilchez-Martinez, et al., Biochem. Biophys. Res. Commun, 59, 1226(1974), for the most part the replacement containing decapeptides havebeen less active.

Now it has been found that the replacement of the glycyl moeity inposition 6 of LH-RH with D-tryptophan gives a compound that is much moreactive and longer acting than LH-RH. Such attributes of the presentdecapeptide have practical significance: the lesser minimum effectivedose reducing side effects as well as the cost for the preparation ofthe compound and the longer acting property reducing the need forfrequent administration.

SUMMARY OF THE INVENTION

The compounds of this invention are selected from the group consistingof (pyro-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂ (1) or anon-toxic, pharmaceutically acceptable salt thereof, and R⁶-(pyro)-Glu-His(N^(1m) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G)-R²)-Pro-Gly-R¹ (2), in which R¹ is selected from the group consistingof amino and 0- (lower alkyl), R², R³, R⁴ and R⁵ are protective groupscapable of being removed by one or more chemical treatments which do notaffect (pyro)-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂ and R⁶ ishydrogen or said protective group.

With reference to R⁶ -(pyro)-Glu-His(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-R¹, in apreferred embodiment R¹ is as defined herein, R² is a protective groupfor the N.sup..sup.δ, N.sup..sup.ω, and N.sup..sup.ω^(') nitrogen atomsof arginine selected from the group consisting of tosyl, nitro,benzyloxycarbonyl and adamantyloxycarbonyl; R³ is a protective group forthe hydroxyl of tyrosine selected from the group consisting of2-bromo-benzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl,tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl;R⁴ is a protective group for the hydroxy group of serine and is selectedfrom the group defined hereinbefore for R³ ; R⁵ is a protective groupfor the imidazole nitrogen atoms of histidine selected from the group oftosyl, dinitrophenyl, 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-1-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group selected from the group consisting oft-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxycarbonyl and d-isobornyl-oxycarbonyl.

A further aspect of the present invention relates to intermediateslinked to a solid resin support. These intermediates are represented bythe formulae:

R⁶ -(pyro)-Glu-His-(N^(1m) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G)-R²)-Pro-Gly-A,

R⁷ -his-(N^(1m) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G)-R²)-Pro-Gly-A,

R⁷ -trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A,

R⁷ -ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A,

R⁷ -tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A and

R⁷ -d-trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A

in which R², R³, R⁴ R⁵ and R⁶ are as defined herein and R⁷ is an α-aminoprotective group known to be useful in the art for the stepwisesynthesis of polypeptides, suitable group being listed hereinafter, andA is an anchoring bond used in solid phase synthesis linked to a solidresin support. A is selected from the class consisting of: ##STR1##

DETAILS OF THE INVENTION

The term "lower alkyl" contemplates alkyl radicals containing from oneto three carbon atoms and includes methyl, ethyl, propyl and isopropyl.

N^(G) means the side chain nitrogen atoms of arginine.

N^(1m) means the imidazole nitrogen atoms of histidine.

The symbol φ means "phenyl".

In general the abbreviations used herein for designating the amino acidsand the protective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomeclature, see Biochemistry 11, 1726 (1972).For instance, t-Boc represents t-butyloxycarbonyl, Z representsbenzyloxycarbonyl, Tos represents tosyl, 2-Br-Cbz represents2-bromo-benzyloxycarbonyl and Bzl represents benzyl. The abbreviationsused herein for the various amino acids are Arg, arginine; Gly, glycine;His, histidine; Leu, Leucine; Pro, proline; (pyro)-Glu,5-oxoproline(pyroglutamic acid); Ser. serine; Trp, tryptophan; and Tyr,tyrosine. All amino acids described herein are in the L-series unlessstated otherwise, i.e., D-Trp is a D-tryptophyl residue.

The polypeptide of this invention can be obtained in the form of an acidaddition salt. Examples of salts are those with organic acids, e.g.acetic, lactic, succinic, benzoic, salicylic, methanesulfonic ortoluenesulfonic acid, as well as polymeric acids such as tannic acid orcarboxymethyl cellulose, and salts with organic acids such as hydrohalicacids, e.g. hydrochloric acid, or sulfuric acid, or phosphoric acid. Ifdesired a particular acid addition salt is converted into another acidaddition salt, e.g. a salt with a non-toxic, pharmaceutically acceptableacid, by treatment with the appropriate ion exchange resin in the mannerdescribed by R. A. Boissonnas, et al., Helv. Chim. Acta, 43, 1349(1960). Suitable ion exchange resins are cellulose based cationexchangers, for example carboxymethylcellulose or chemically modified,cross linked dextran cation exchangers, for example, those of thesephadex C-type, and strongly basic anion exchange resins, for examplethose listed by J.P. Greenstein and M. Winitz in "Chemistry of the AminoAcids", John Wiley and Sons, Inc., New York and London, 1961, Vol. 2, p.1456.

The decapeptide of this invention and its salts possess valuable,long-acting LH- and FSH-releasing hormone activity.

The valuable LH- and FSH-releasing hormone activity and long actingproperty of the compound of this invention are demonstrated by standardpharmacological procedures. For example, these activities can bedemonstrated by tests described by A. Arimura, et al., Endocrinology,95, 1174 (1974). More specifically, by following the procedure describedtherein, LH-release data obtained from rats given equal doses (50 ng,subcutaneously) show that [D-Trp⁶ ]-LH-RH reaches peak activity at abouttwo hours after dosing and that significant activity is still present upto 6 hours; whereas after an injection of LH-RH, peak activity isreached at about the 15 minute mark and no effects of the injection areobserved after one hour. Also integrated levels of LH over a 6 hourperiod indicate that [D-Trp⁶ ]-LH-RH is about 12 times more active inreleasing LH-RH. FSH-release data following injections of the twocompounds indicate that ]D-Trp⁶ ]-LH-RH is about 20 times more activethan LH-RH at the same dose (50 ng).

Moreover, the activity of the compound of this invention is demonstratedreadily in man.

Radioimmunoassay of serum levels of LH after intranasal administrationindicates that the minimum effective dose of LH-RH is about 2.0 mgwhereas an equivalent degree of activity is obtained with a 0.5 mg doseof [D-Trp⁶ ]-LH-RH. In this instance the compounds are given to humansin a normal saline solution. With regard to FSH release in mancomparative studies with LH-RH and [D-Trp⁶ ]-LH-RH give particularlynoteworthy results. Intranasal administration of up to 2.0 mg of LH-RHhas little or no effect on FSH serum levels as measured byradioimmunoassay, H. G. Dahlin, et al., Horm. Metab. Res., 6, 510(1974); however, the compound of this invention releases significantamounts of FSH, i.e., levels ranging from 0.3 to greater than 1.5miu/ml, at 0.5 mg under the same conditions. It will be readilyappreciated that a compound that is able to effectively release FSH hasmany therapeutic applications; see, for example, H.G. Dahlen, et al.,cited above.

The LH- and FSH-releasing properties of the compound of this invention,which in turn induce ovulation in animals, make the hormone useful inveterinary practice and in animal husbandry. It is often desirable tosynchronize estrus in livestock, for example, cattle, sheep or swine,either in order to be able to mate all the females in a given group witha male of the desired genetic quality, or so as to be able to performartificial insemination on a maximum number of females, both within acomparatively short period of time. In the past, this has been done byadministering to the animals and ovulation-inhibiting agent, withdrawingadministration of said agent shortly before the date chosen for matingor artificial insemination, and relying either upon the naturalproduction of LH and FSH to induce ovulation and to produce estrus or byadministering gonadotrophins. However, this procedure was not entirelysatisfactory because ovulation at a predetermined time occurred never inall the animals together but only in a certain proportion thereof whengonadotrophins were not used. On the other hand, the high cost ofgonadotrophins and side effects encountered in their adminstration madethis method impractical. It is now possible to obtain substantiallycomplete synchronization of ovulation and of estrus, by treating theanimals in a given group first with an ovulation inhibitor which issubsequently withdrawn, and then administering ]D-Trp⁶ ]-LH-RH shortlybefore the predetermined period of time for mating or artificialinsemination, so as to obtain ovulation and estrus within that timeinterval. The delay in the onset of ovulation and estrus followingadministration of [D-Trp⁶ ]-LH-RH varies with the species of animals,and the optimal time interval has to be chosen for each species. Forexample, in rodents such as rats or hamsters ovulation takes placewithin 18 hours following administration of the decapeptide of thisinvention.

The method described above for obtaining ovulation and estrus within aprecisely predetermined time interval, so as to be certain of asuccessful mating, is particularly important for breeders of race horsesand of show animals, where the fees paid for the services of anexceptional male animal often amount to very considerable sums of money.[D-Trp⁶ ]-LH-RH is also useful to increase the number of live births perpregnancy in livestock, for example, cattle, sheep or swine. For thispurpose the decapeptide is given in a series of parenteral doses,preferably by intravenous or subcutaneous injections, in the range of0.1 -10 mcg. per kilogram of body weight per day, 96 to 12 hours priorto expected estrus and subsequent mating. A priming injection of 1000 to5000 iu of pregnant mares serum gonadotrophin may also be given one tofour days prior to the above injection of the decapeptide. A similartreatment, with or without prior priming, is also useful for inducingpuberty in farm animals.

When the decapeptide is employed for the purpose of inducing ovulationand estrus or for inducing puberty in warm-blooded animals, especiallyin rodents such as rats or hamsters or in livestock it is administeredsystemically, preferably parenterally, in combination with apharmaceutically acceptable liquid or solid carrier. The proportion ofthe decapeptide is determined by its solubility in the given carrier, bythe chosen route of adminstration, and by standard biological practice.For parenteral administration to animals the decapeptide is used in asterile aqueous solution which may also contain other solutes such asbuffers or preservatives, as well as sufficient pharmaceuticallyacceptable salts or glucose to make the solution isotonic. The dosagewill vary with the form of administration and with the particularspecies of animal to be treated and is preferably kept at a level offrom 0.1 mcg. to 10 mcg. per kilogram body weight. However, a dosagelevel in the range of from about 1 mcg. to about 5 mcg. per kilogrambody weight is most desirably employed in order to achieve effectiveresults.

The decapeptide may also be administered in one of the long-acting,slow-release or depot dosage forms described below, preferably byintramuscular injection or by implantation. Such dosage forms aredesigned to release from about 0.1 mcg. to about 10 mcg. per kilogrambody weight per day. [D-Trp⁶ ]-LH-RH is also useful in human medicine.For example, human chorionic gonadotrophin (HCG) which contains mainlyLH and some FSH has been used for over 30 years to treat certainendocrinological disorders such as disturbances of the cycle,amenorrhea, lack of development of secondary sec characteristics, andinfertility in the female, or certain cases of hypogonadism delayedpuberty, cryptorchidism, and non-psychogenic impotence in the male.Lately, infertility in the human female has also been treated with humanmenopausal gonadotrophin (HMG) which contains mainly FSH, followed bytreatment with HCG. One of the disadvantages of the treatment ofinfertility in the human female with HCG or with HMG followed by HCG hadbecome apparent in that such treatment often results in superovulationand unwanted multiple births, probably because of the impossibility ofgiving only the exact amounts of FSH and LH which are necessary forovulation. The administration of the decapeptide of this inventionovercomes the above disadvantage, because the compound causes release ofLH and FSH by the pituitary only in the exact quantities which arerequired for normal ovulation. For that reason the decapeptide of thisinvention is not only useful for the above purpose, but it is equallyuseful in the human female in the treatment of disturbances of thecycle, of amenorrhea, of hypogonadism, and of lack of development ofsecondary sex characteristics.

Furthermore, the decapeptide of this invention is useful incontraception. For example, when the decapeptide is administered to ahuman female early in the menstrual cycle LH is released at that timeand causes premature ovulation. The immature ovum is either not capableof being fertilized, or, if fertilization should nevertheless have takenplace, it is highly unlikely that the fertilized ovum will becomeimplanted because the estrogen-progestin balance required to prepare theendometrium is not present and the endometrium is not in the conditionnecessary for implantation. On the other hand, when the decapeptide isadminstered towards the end of the cycle the endometrium is disruptedand menstruation takes place.

In addition, the decapeptide of this invention is also useful incontraception by the "rhythm" method, which has always been relativelyunreliable because of the impossibility of predetermining ovulation inthe human female with the required degree of accuracy. Administration ofthe decapeptide at mid-cycle, i.e. at about the normally expected timefor ovulation, induces ovulation shortly thereafter and makes the rhythmmethod both safe and effective.

The decapeptide is also useful as a diagnostic tool for distinguishingbetween hypothalamic and pituitary malfunctions or lesions in the humanfemale. When administering the decapeptide to a patient suspected ofsuch malfunctions or lesions and a rise in the level of LH issubsequently observed there is good indication to conclude that thehypothalamus is the cause of the malfunction and that the pituitary isintact. On the other hand, when no rise in circulating LH is seenfollowing the administration of the decapeptide a diagnosis of pituitarymalfunction or lesion can be made with a high degree of confidence.

In the human male, administration of decapeptide provides the amounts ofLH (or ICSH) and of FSH necessary for normal sexual development in casesof hypogonadism or delayed puberty, and is also useful in the treatmentof cryptorchidism. Furthermore, the FSH released by the administrationof the decapeptide stimulates the development of germinal cells in thetestes, and the decapeptide is useful in the treatment of psychogenicand nonpsychogenic impotence.

When the decapeptide, preferably in the form of an acid addition salt,is employed in human medicine, it is administered systemically, eitherby intravenous, subcutaneous, or intramuscular injection, or bysublingual, nasal, or vaginal administration, in compositions inconjunction with a pharmaceutically acceptable vehicle or carrier.

For administration by the nasal route as drops or spray it is preferredto use the decapeptide in solution in a sterile aqueous vehicle whichmay also contain other solutes such as buffers or preservatives, as wellas sufficient quantities of pharmaceutically acceptable salts or ofglucose to make the solution isotonic. Doses by the intranasal routerange from 0.1 to 50 mcg/kg, or preferably 0.5 to 10 mcg/kg.

The decapeptide may also be administered as nasal or vaginal powders orinsufflations. For such purposes the decapeptide is adminstered infinely divided solid form together with a pharmaceutically acceptablesolid carrier, for example a finely divided polyethylene glycol("Carbowax 1540"), finely divided lactose, or preferably for vaginaladministration, very finely divided silica ("Cab-O-Sil"). Suchcompositions may also contain other excipients in finely divided solidform such as preservatives, buffers, or surface active agents.

For sublingual or vaginal administration it is preferred to formulatethe decapeptide in solid dosage forms such as sublingual tablets orvaginal inserts or suppositories with sufficient quantities of solidexcipients such as starch, lactose, certain types of clay, buffers, andlubricating, disintegrating, or surface-active agents, or withsemi-solid excipients commonly used in the formulation of suppositories.Examples of such excipients are found in standard pharmaceutical texts,e.g. in Remington's Pharmaceutical Sciences, Mack Publishing Company,Easton, Pa., 1970.

The dosage of the decapeptide will vary with the form of administrationand with the particular patient under treatment. Generally, treatment isinitiated with small dosages substantially less than the optimum dose ofthe compound. Thereafter, the dosage is increased by small incrementsuntil the optimum effect under the circumstances is reached. In general,the decapeptide obtained by the process is most desirably administeredat a concentration level that will generally afford effective release ofLH and of FSH without causing any harmful or deleterious side effects,and preferably at a level that is in a range of from about 0.1 mcg. toabout 100 mcg. per kilogram body weight, although as aforementionedvariations will occur. However, a dosage level that is in the range offrom about 0.5 mcg to about 5 mcg per kilogram body weight is mostdesirably employed in order to achieve effective results.

It is often desirable to administer the decapeptide continuously overprolonged periods of time in long-acting, slow-release, or depot dosageforms. Such dosage forms may either contain a pharmaceuticallyacceptable salt of the compound having a low degree of solubility inbody fluids, for example one of those salts described below, or they maycontain the decapeptide in the form of a water-soluble salt togetherwith a protective carrier which prevents rapid release. In the lattercase, for example, the decapeptide may be formulated with anon-antigenic partially hydrolyzed gelatin in the form of a viscousliquid; or it may be absorbed on a pharmaceutically acceptable solidcarrier, for example zinc hydroxide, and may be administered insuspension in a pharmaceutically acceptable liquid vehicle; or thedecapeptide may be formulated in gels or suspensions with a protectivenon-antigenic hydrocolloid, for example sodium carboxymethylcellulose,polyvinylpyrrolidone, sodium alginate, gelatine, polygalacturonic acids,for example, pectin, or certain mucopolysaccharides, together withaqueous or nonaqueous pharmaceutically acceptable liquid vehicles,preservatives, or surfactants. Examples of such formulations are foundin standard pharmaceutical texts, e.g. in Remington's PharmaceuticalSciences, cited above. Long-acting, slow-release preparations of thedecapeptide may also be obtained by microencapsulation in apharmaceutically acceptable coating material, for example gelatine,polyvinyl alcohol or ethyl cellulose. Further examples of coatingmaterials and of the processes used for microencapsulation are describedby J. A. Herbig in "Encyclopedia of Chemical Technology", Vol. 13, 2ndEd., Wiley, New York, 1967, pp. 436-456. Such formulations, as well assuspensions of salts of the decapeptide which are only sparingly solublein body fluids, are designed to release from about 0.1 mcg to about 50mcg of the hormone per kilogram body weight per day, and are preferablyadministered by intramuscular injection. Alternatively, some of thesolid dosage forms listed above, for example certain sparinglywater-soluble salts or dispersions in or adsorbates on solid carriers ofsalts of the decapeptide, for example dispersions in a neutral hydrogelof a polymer of ethylene glycol methacrylate or similar monomerscross-linked as described in U.S. Pat. No. 3,551,556 may also beformulated in the form of pellets releasing about the same amounts asshown above and may be implanted subcutaneously or intramuscularly.

Alternatively, slow-release effects over prolonged periods of time mayalso be obtained by administering the decapeptide obtained by theprocess of this invention as an acid addition salt in an intra-vaginaldevice or in a temporary implant, for example a container made of anon-irritating silicone polymer such as a polysiloxane, e.g. "Silastic",or of a neutral hydrogel of a polymer as described above, possessing therequired degree of permeability to release from about 0.1 mcg. to about50 mcg per kilogram body weight per day. Such intra-vaginal or implantdosage forms for prolonged administration have the advantage that theymay be removed when it is desired to interrupt or to terminatetreatment.

PROCESS

In selecting a particular side chain protective group to be used in thesynthesis of the present decapeptide, the following rules should befollowed: (a) the protective group must be stable to the reagent andunder the reaction conditions selected for removing the α-aminoprotective group at each step of the synthesis, (b) the protective groupmust retain its protecting properties (i.e., not be split off undercoupling conditions), and (c) the side chain protective group must beremovable upon the completion of the synthesis containing the desiredamino acid sequence under reaction conditions that will not alter thepeptide chain.

With reference to R⁷, suitable protective groups include (1) aliphaticurethan protective groups illustrated by t-butyloxycarbonyl,diisopropylmethoxycarbonyl, biphenylisopropyloxycarbonyl,isopropyloxycarbonyl, t-amyloxycarbonyl, ethoxycarbonyl,allyloxycarbonyl; (2) cycloalkyl urethan type protective groupsillustrated by cyclopentyloxycarbonyl, adamantyloxycarbonyl,d-isobornyloxycarbonyl, cyclohexyloxycarbonyl; nitrophenylsulfenyl,tritylsulfenyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl and trityl.The preferred α-amino protective group for R⁷ are selected from thegroup consisting of t-butyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxycarbonyl, d-isobornyloxycarbonyl, o-nitrophenylsulfenyl,biphenylisopropyloxycarbonyl, and α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl.

The decapeptide of this invention is prepared using solid phasesynthesis. The synthesis is commenced from the C-terminal end of thepeptide using an α-amino protected resin. Such a starting material isprepared by attaching an α-amino protected glycine to a benzhydrylamineresin, a chloromethylated resin or a hydroxymethyl resin, the formerbeing preferred. The preparation of a benzhydrylamine resin is describedby P. Rivaille, et al., Helv. Chim. Acta, 54,2772 (1971) and thepreparation of the hydroxymethyl resin is described by M. Bodanszky andJ. T. Sheehan, Chem. Ind (London) 38, 1597 (1966). A chloromethylatedresin is commercially available from Bio Rad Laboratories, Richmond,California. In using the benzhydrylamine resin an amide anchoring bondis formed with the α-amino protected glycine as follows: ##STR2##

This permits the C-terminal amide function to be obtained directly afterthe amino acid sequence in the synthesis is completely cleaving off theresin support of the linked peptide to form the glycine amide at theC-terminal portion of the desired decapeptide. In this instance the useof hydrogen fluoride for cleaving off the resin support also removes theside chain protective groups to give the decapeptide of this invention.

When the other resins are used, the anchoring bond is the benzylestergroup as illustrated hereinbefore. In this instance a convenientprocedure for converting the linked protected peptide to the C-terminalamide is to ammonolize the protected peptide off the resin and thenremove the protective groups of the resulting amide by treatment withsodium and liquid ammonia or by hydrogen fluoride cleavage. Analternative procedure would be to cleave by transesterification with alower alkanol, preferably methanol or ethanol, in the presence oftriethylamine and then convert the resulting ester into an amide andsubsequently deprotect as described above. See also J. M. Steward and J.D. Young, "Solid Phase Peptide Synthesis", W. H. Freeman & Co., SanFrancisco, 1969, pp. 40-49.

More specifically, in an embodiment of the present invention an α-aminoprotected glycine, preferably t-butyloxycarbonylglycine, is coupled tobenzhydrylamine resin with the aid of the carboxyl group activatingcompound, preferably, dicyclohexylcarbodiimide. Following the couplingof the α-amino protected glycine to the resin support, the α-aminoprotecting group is removed such as by using trifluoroacetic acid inmethylene chloride, trifluoroacetic acid alone or hydrochloric acid indioxane. The deprotection is carried out at a temperature between about0° C and room temperature. Other standard cleaving reagents andconditions for removal of specific α-amino protecting groups may be usedas described by E. Schroder and K. Lubke, "The Peptides", Vol. 1,Academic Press, New York, 1965, pp. 72-75. After removal of the α-aminoprotecting group, the remaining α-amino protected amino acids arecoupled step-wise in the desired order to obtain the decapeptide. Eachprotected amino acid is introduced into the solid phase reactor in abouta three-fold excess and the coupling is carried out in a medium ofmethylene chloride or mixtures of dimethylformamide in methylenechloride. In cases where incomplete coupling occurred the couplingprocedure is repeated before removal of the α-amino protecting group,prior to the coupling of the next amino acid to the solid phase reactor.The success of the coupling reaction at each stage of the synthesis ismonitored by the ninhydrin reaction as described by E. Kaiser, et al.,Analyt. Biochem. 34, 595 (1970).

After the desired amino acid sequence has been synthesized, the peptideis removed from the resin support by treatment with a reagent such ashydrogen fluoride which not only cleaves the peptide from the resin butalso cleaves all remaining side chain protecting groups and the α-aminoprotecting group (if present) on the pyroglutamic acid residue to obtaindirectly the decapeptide in the case where the benzhydrylamine resin wasused.

Where a chloromethylated resin is used the peptide may be separated fromthe resin by transesterification with a lower alkanol, preferablymethanol or ethanol, after which the recovered product ischromatographed on silica gel and the collected fraction subjected totreatment with ammonia to convert the lower alkyl ester, preferably themethyl or ethyl ester, to the C-terminal amide. The side chainprotecting groups are then cleaved by procedures described above, forexample by treatment with sodium in liquid ammonia or by hydrogenfluoride.

Although a solid phase synthesis of the decapeptide of this invention isdisclosed herein, the preparation of the decapeptide also can berealized by classical methods. For example, by following the procedureof H. U. Immer et al., U.S. Pat. No. 3,853,108, issurd September 10,1974 but substituting Ser-Tyr-D-Trp-NHNHBoc for Ser-Tyr-Gly-NHNHBoc, thedecapeptide also is obtained (via(pyro)-Glu-His-Trp-Ser-Tyr-D-Trp-NHNH₂).

The following Examples illustrate further this invention.

EXAMPLE 1

L-Pyroglutamyl-L-histidyl(tosyl)-L-tryptophyl-L-seryl(benzyl-L-tyrosyl(2-bromo-benzyloxycarbonyl)-D-tryptophyl-L-leucyl-L-arginyl(tosyl)-L-prolylglycylbenzhydrylamineresin (R⁶ -(pyro)-Glu-His-(N^(Im)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A; R² = Tos,R³ = 2-Br-Cbz, R⁴ = Bzl, R⁵ = Tps, R⁶ = H and A = benzhydrylamineresin).

Benzhydrylamine resin (1.25 g, 1.0 mmole) is placed in the reactionvessel of a Beckman Model 990 automatic peptide synthesizer programmedto carry out the following wash cycle: (a) methylene chloride; (b) 33%trifluoroacetic acetic in methylene chloride (2times for 2.5 and 25minutes each); (c) methylene chloride; (d) ethanol; (e) chloroform; (f)10% triethylamine in chloroform (2 times for 25 minutes each); (g)chloroform; (h) methylene chloride.

The washed resin is then stirred with t-butyl-oxycarbonyl glycine (525mg, 3.0 mmoles) in methylene chloride and dicyclohexylcarbodiimide (3.0mmoles) is added. The mixture is stirred at room temperature (22°-25° C)for 2 hours and the amino acid resin is then washed successively withmethylene chloride (3 times), ethanol (3 times), and methylene chloride(3 times). The attached amino acid is deprotected with 33%trifluoroacetic acid in methylene chloride (2 times for 2.5 and 25minutes each and then steps (c) through (h) as described in the abovewash cycle are performed.

The following amino acids (3.0 mmoles) are then coupled successively bythe same cycle of events: t-Boc-L-proline; t-Boc-L-arginine(Tos);t-Boc-L-leucine; t-boc-D-tryptophan; t-boc-L-tyrosine (2-Br-Cbz);t-Boc-L-serine(Bzl); t-Boc-L-tryptophan; t-Boc-L-histidine(Tos);L-(pyro)-glutamic acid.

The completed decapeptide resin is washed with methylene chloride (3times) followed by methanol (3 times) and dried under reduced pressurewhereupon 98 % of the theoretical weight gain is obtained.

The benzhydrylamine resin used in this example is a commerciallyavailable resin (1% cross linked, Bachem Inc., Marina del Rey,California).

EXAMPLE 2L-Pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide

Removal of protecting groups and cleavage of the decapeptide from theresin is carried out by treatment of 1.0 g of material with hydrogenfluoride (24 ml) and anisole (6 ml) at 0° C for 30 minutes. The hydrogenfluoride is removed under reduced pressure and the anisole removed bywashing with ethyl acetate.

The crude peptide is purified by gel filtration on a column (2.5 × 100cm) of Sephadex G-25 (a fine grade, chemically modified cross-linkeddextran) by elution with 2 molar acetic acid and fractions shown tocontain a major peak by UV absorption at 280 nm were pooled andevaporated to dryness.

The residual oil was applied to a column (2.5 × 100 cm) of Sephadex G-25(fine), previously equilibrated with the lower phase followed by theupper phase of n-butanol; acetic acid; water (4:1:5) solvent system.Elution with the upper phase gives a major peak and material from thisarea was subjected to elution on a column (1.4 × 94 cm) of carboxymethylcellulose according to the conditions described by D. H. Coy, et al., J.Med. Chem., 16, 1140 (1973). Appropriate fractions (1050 -1190 ml),after lyophilization to constant weight, gave D-Trp⁶ -LH-RH as a white,fluffy powder (80 mg); [α]_(D) ²³ -58.8° (c = 0.33, IN HOAc).

The product was homogeneous by thin layer chromatography in fourseparate solvent systems when loads of 20-30 mcg were applied and spotsvisualized by exposure to iodine vapour followed by Ehrlich reagent. Thefollowing Rf values were obtained:

1-butanol: acetic acid: water (4:1:5: upper phase), 0.25; ethyl acetate:pyridine: acetic acid: water (5:5:1:3), 0.63: 2-propanol: 1 M aceticacid (2:1), 0.38; 1-butanol: acetic acid: water: ethyl acetate(1:1:1:1), 0.51.

Amino acid analysis gave: Glu, 1.08; His, 0.95; Trp, 2.00; Ser, 0.94;Tyr, 0.97; Leu, 0.93; Arg, 0.98; Pro, 1.00; Gly, 1.02; NH₃, 1.03.

We claim:
 1. A compound of the formula R⁶ -(Pyra)-Glu-His(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-R¹ in which R¹is amino or 0-(lower alkyl), R² is a protective group for the N.sup.δ ,N.sup.ω , and N.sup.ω^(') nitrogen atoms of arginine selected from thegroup consisting of tosyl, nitro, benzyloxycarbonyl andadamantyloxycarbonyl;R³ is a protective group for the hydroxyl oftyrosine selected from the group consisting of 2-bromobenzyloxycarbonyl,benzyl, acetyl, tosyl, benzoyl, t-butyl, tetrahydropyran-2-yl, trityl,2,4-dichlorobenzyl and benzyloxycarbonyl; R⁴ is a protective group forthe hydroxyl group of serine and is selected from the group definedhereinbefore for R³ ; R⁵ is a protective group for the imidazolenitrogen atoms of histidine selected from the group of dinitrophenyl,tosyl, 2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group selected from the group consisting oft-butyloxycarbonyl, benzyloxycarbonyl, cyclopentyloxycarbonyl,t-amyloxy-carbonyl and d-isobornyloxycarbonyl.
 2. The compound of claim1 in which R¹ is amino.
 3. The compound of claim 1 wherein R¹ is amino,R² is tosyl, R³ is 2-bromo-benzyloxycarbonyl, R⁴ is benzyl, R⁵ is tosyland R⁶ is hydrogen.
 4. The compound which isL-pyroglutamyl-L-histidyl-L-tryptophyl-L-seryl-L-tyrosyl-D-tryptophyl-L-leucyl-L-arginyl-L-prolylglycinamide,or a non-toxic, pharmaceutically acceptable addition salt thereof.
 5. Acompound selected from the group consisting of R⁶-(pyro)-Glu-His-(N^(lm) -R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G)-R²)-Pro-Gly-A, R⁷ -His(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr-(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Trp-Ser(R⁴)-tyr-(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Ser(R⁴)-Tyr-(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A, R⁷-Tyr(R³)-D-Trp-Leu-Arg-(N^(G) -R²)-Pro-Gly-A, and R⁷-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A, in which [R², R³, R⁴ and R⁵ areprotective groups capable of being removed by one or more chemicaltreatments which do not effect(pyro)-Glu-His-Trp-Ser-Tyr-D-Trp-Leu-Arg-Pro-Gly-NH₂,] R² is aprotective group for the N.sup.σ , N.sup.ω , and N.sup.ω nitrogen atomsof arginine selected from the group consisting of tosyl, nitro,benzyloxycarbonyl and adamantyloxycarbonyl;R³ is a protective group forthe hydroxyl of tyrosine selected from the group consisting of2-bromo-benzyloxycarbonyl, benzyl, acetyl, tosyl, benzoyl, t-butyl,tetrahydropyran-2-yl, trityl, 2,4-dichlorobenzyl and benzyloxycarbonyl;R⁴ is a protective group for the hydroxyl group of serine and isselected from the group defined hereinbefore for R³ ; R⁵ is a protectivegroup for the imidazole nitrogen atoms of histidine selected from thegroup of dinitrophenyl, tosyl,2,2,2-trifluoro-1-benzoyloxycarbonylaminoethyl and2,2,2-trifluoro-t-butyloxycarbonylaminoethyl; and R⁶ is hydrogen or anα-amino protective group and R⁷ is an α-amino protective group, and A isselected from the class consisting of: ##STR3##
 6. The compoundaccording to claim 5 in which A is a benzhydrylamine resin.
 7. Thecompound of claim 5 having the formula R⁶ -(pyro)-Glu-His-(N^(lm)-R⁵)-Trp-Ser(R⁴)-Tyr(R³)-D-Trp-Leu-Arg(N^(G) -R²)-Pro-Gly-A in which R²is tosyl, R³ is 2-bromo-benzyloxycarbonyl, R⁴ is benzyl, R⁵ is tosyl, R⁶is hydrogen and A is benzhydrylamine resin.